Telomere loss, senescence, and genetic instability in CD4+ T lymphocytes overexpressing hTERT

Little is known about the long-term consequences of overexpression of the human telomerase reverse transcriptase (hTERT) gene in T lymphocytes. To address this issue, we transduced polyclonal as well as clonally derived populations of naive and memory CD4+ T cells from 2 healthy donors (aged 24 and 34 years) with retroviral vectors encoding green fluorescence protein (GFP) and hTERT (GFP-hTERT) or GFP alone. After transduction, cells were sorted on the basis of GFP expression and cultured in vitro until senescence. T cells transduced with hTERT exhibited high stable telomerase activity throughout the culture period. Relative to GFP controls, minor changes in overall gene expression were observed yet the proliferative lifespan of the hTERT-transduced populations was significantly increased and the rate of telomere loss was lower. Nevertheless, hTERT-transduced cells showed progressive telomere loss and had shorter telomeres at senescence than controls (2.3 ± 0.3 kilobase [kb] versus 3.4 ± 0.1 kb). Furthermore, a population of cells with 4N DNA consisting of binucleated cells with connected nuclei emerged in the hTERT-transduced cells prior to senescence. We conclude that overexpression of hTERT in CD4+ T cells provides a proliferative advantage independent of the average telomere length but does not prevent eventual genetic instability and replicative senescence.

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Cisplatin-induced apoptotic endonuclease EndoG inhibits telomerase activity and causes malignant transformation of human CD4+ T lymphocytes

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20176301013 ◽

2017 ◽

Vol 63 (1) ◽

pp. 13-26 ◽

Cited By ~ 5

Author(s):

D.D. Zhdanov ◽

D.A. Vasina ◽

E.V. Orlova ◽

V.S. Orlova ◽

V.S. Pokrovsky ◽

...

Keyword(s):

T Cells ◽

Alternative Splicing ◽

Malignant Transformation ◽

Cd4 T Cells ◽

Replicative Senescence ◽

Telomerase Activity ◽

Full Length ◽

Leukemic Cells ◽

Proliferative Potential ◽

Human Telomerase Reverse Transcriptase

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.

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Influenza virus-specific CD4+ T helper type 2 T lymphocytes do not promote recovery from experimental virus infection.

Journal of Experimental Medicine ◽

10.1084/jem.180.4.1273 ◽

1994 ◽

Vol 180 (4) ◽

pp. 1273-1282 ◽

Cited By ~ 196

Author(s):

M B Graham ◽

V L Braciale ◽

T J Braciale

Keyword(s):

Immune Response ◽

T Cells ◽

T Lymphocytes ◽

Cd4 T Cells ◽

Primary Role ◽

T Helper ◽

Helper Type

T lymphocytes play a primary role in recovery from viral infections and in antiviral immunity. Although viral-specific CD8+ and CD4+ T cells have been shown to be able to lyse virally infected targets in vitro and promote recovery from lethal infection in vivo, the role of CD4+ T lymphocytes and their mechanism(s) of action in viral immunity are not well understood. The ability to further dissect the role that CD4+ T cells play in the immune response to a number of pathogens has been greatly enhanced by evidence for more extensive heterogeneity among the CD4+ T lymphocytes. To further examine the role of CD4+ T cells in the immune response to influenza infection, we have generated influenza virus-specific CD4+ T cell clones from influenza-primed BALB/c mice with differential cytokine secretion profiles that are defined as T helper type 1 (Th1) clones by the production of interleukin 2 (IL-2) and interferon gamma (IFN-gamma), or as Th2 clones by the production of IL-4, IL-5, and IL-10. Our studies have revealed that Th1 clones are cytolytic in vitro and protective against lethal challenge with virus in vivo, whereas Th2 clones are noncytolytic and not protective. Upon further evaluation of these clonal populations we have shown that not only are the Th2 clones nonprotective, but that pulmonary pathology is exacerbated as compared with control mice as evidenced by delayed viral clearance and massive pulmonary eosinophilia. These data suggest that virus-specific CD4+ T cells of the Th2 subset may not play a primary role in virus clearance and recovery and may lead to immune mediated potentiation of injury.

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Telomere Length, Telomerase Activity, and Replicative Potential in HIV Infection: Analysis of CD4+ and CD8+T Cells from HIV-discordant Monozygotic Twins

Journal of Experimental Medicine ◽

10.1084/jem.185.7.1381 ◽

1997 ◽

Vol 185 (7) ◽

pp. 1381-1386 ◽

Cited By ~ 91

Author(s):

Larry D. Palmer ◽

Nan-ping Weng ◽

Bruce L. Levine ◽

Carl H. June ◽

H. Clifford Lane ◽

...

Keyword(s):

T Cells ◽

Hiv Infection ◽

Telomere Length ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Replicative Senescence ◽

Monozygotic Twins ◽

Telomerase Activity ◽

Replicative Capacity

To address the possible role of replicative senescence in human immunodeficiency virus (HIV) infection, telomere length, telomerase activity, and in vitro replicative capacity were assessed in peripheral blood T cells from HIV+ and HIV− donors. Genetic and age-specific effects on these parameters were controlled by studying HIV-discordant pairs of monozygotic twins. Telomere terminal restriction fragment (TRF) lengths from CD4+ T cells of HIV+ donors were significantly greater than those from HIV− twins. In contrast, telomere lengths in CD8+ T cells from HIV+ donors were shorter than in HIV− donors. The in vitro replicative capacity of CD4+ cells from HIV+ donors was equivalent to that of HIV− donors in response to stimulation through T cell receptor CD3 and CD28. Little or no telomerase activity was detected in freshly isolated CD4+ or CD8+ lymphocytes from HIV+ or HIV− donors, but was induced by in vitro stimulation of both HIV+ and HIV− donor cells. These results suggest that HIV infection is associated with alterations in the population dynamics of both CD4+ and CD8+ T cells, but fail to provide evidence for clonal exhaustion or replicative senescence as a mechanism underlying the decline in CD4+ T cells of HIV-infected donors.

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Two Novel WT-1 Derived Peptides Induce CD4+ peptide–specific T Cell Proliferation in Patients with Myelodysplastic Syndrome (MDS)

Blood ◽

10.1182/blood.v112.11.5432.5432 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5432-5432

Author(s):

Monica Bocchia ◽

Micaela Ippoliti ◽

Marzia Defina ◽

Rosaria Crupi ◽

Maristella Tassi ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Cytotoxic Activity ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

T Cell Proliferation ◽

Wt1 Protein

Abstract The Wilms tumor gene WT1 is overexpressed in hematopoietic malignancies such as Myelodysplastic syndromes and leukemias and the WT1 protein was demonstrated to be an attractive target antigen for an immunotherapeutic approach to these diseases. Most of the efforts have been focused to the search for immunogenic peptides suitable for inducing cytotoxic T lymphocytes (CTLs) and to less extent for CD4+ T lymphocytes with potential cytotoxic activity. On this matter, in our previous experience with a p210-derived peptide vaccine developed for chronic myeloid leukemia patients with minimal residual disease, the main immune and therapeutic effect observed after vaccinations appeared to be mediated by peptide-specific CD4+ T cells induced by the longest peptide (25 mer) included in our vaccine. CML-peptide specific T cells were found to be either CD4+/perforin+ or CD4+/CD25+/Foxp3+ and we recently showed their direct cytotoxicity against a CML cell line. Thus to pursue a vaccine strategy mainly devoted to a similar CD4+ T cell immune response, we screened WT1 protein through Syfpeithi database to identify original peptides with a suitable length (23–25 amino acids) to be processed by several HLA class II molecules and to induce a strong CD4+ T cell stimulation. Additionally, in order to maximize the immunogenic potential of the novel peptides, we focused our attention on areas of the protein with known CTLs/CD4 T cells immunogenic epitopes. We identified two peptides that fulfilled these requirements: SEPQQMGSDVRDLNALLPAVPSLGG (WT1-iso5 64–88) which includes 5 amino acid from the alternative splicing derived isoform 5 of WT1 and the first 20 aa of “canonical” WT1 sequence and RPFMCAYPGCNKRYFKLSHLQMHSR (WT1321–345). Both 25mer peptides showed strong HLA binding properties for HLA-DRB1*0101, HLADRB1* 0401, HLA-DRB1*0701, HLA-DRB1*1101, HLA-DRB1*1501 and HLADRB1* 0301( DR17). We first tested them in vitro for their capability to induce peptide-specific CD4+ T cells. Briefly, CD4+ T cells freshly isolated from PBMC were cultured for 21 days in 5% AB human serum media while undergoing to 3 rounds of stimulation with autologous CD14+ cells and both WT1-iso5 64–88 and WT1 321–345 peptides at 20μg/ml in the presence of IL-15. This in vitro stimulation was performed in 3 normal subjects and in 3 MDS patients with high levels of bone marrow WT1 transcript (2 patients presenting a low-International Prognostic Scoring System (IPSS) refractory anemia (RA) and 1 with intermediate IPSS RA). In all 3 healthy donors tested, both peptides were able to induce peptide specific CD4+ T cell proliferation as measured by standard 3HThymidine assay, with a stimulation index (SI) ranging from 2.0 to 2.5 regardless of their HLA-DR phenotype ( SI= cpm CD4+ T cells plus test peptides/CD4+ T cell alone or CD4+ T cells plus control peptides; peptide-specific T cell proliferation was considered positive for SI≥2). Similar results were obtained in all 3 MDS patients in which WT1-iso5 64–88 and WT1 321–345 induced peptide-specific CD4+ T cell proliferation with a SI value of 2.5, 2.9 and 3.0 respectively. In conclusion the present study identified 2 novel WT1-derived 25 mer peptides which were able to easily induce in vitro a peptide-specific CD4+ T cell response in MDS patients. WT1-specific CD4+ T cells proliferated with similar SI values in normal donors and in WT1 positive MDS patients, the latter being highly exposed to this antigen and thus potentially tolerant to it. A possible cytotoxic activity of these WT1-specific CD4+ T cells is under evaluation and in vivo vaccinations of low-intermediate IPSS MDS patients with these peptides are planned.

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Possible involvement of autoreactive CD4 T cells in generation of cytotoxic T lymphocytes on in vitro stimulation with H-2 class I- binding tumor antigen peptide

International Immunology ◽

10.1093/intimm/10.8.1167 ◽

1998 ◽

Vol 10 (8) ◽

pp. 1167-1174 ◽

Cited By ~ 1

Author(s):

S Honda

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Tumor Antigen ◽

Cd4 T Cells ◽

Class I ◽

Antigen Peptide

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Modulation of TRPV1 Function in Human CD4+ T Cells by Nanodiamond and Nanoplatinum Liquid, DPV576

10.20944/preprints201804.0354.v1 ◽

2018 ◽

Author(s):

Mamdooh H. Ghoneum ◽

James K. Gimzewski ◽

Aya D. Ghoneum ◽

Hideki Katano ◽

Anshu Agrawal

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Cd4 T Cells ◽

Transient Receptor Potential ◽

Receptor Expression ◽

Transient Receptor Potential Vanilloid ◽

Trpv1 Channels ◽

Short Term Exposure ◽

Trpv Channels

Transient receptor potential vanilloid (TRPV) channels act as sensors of pain, temperature, and other external stimuli. We have recently shown that DPV576, an aqueous mixture of nanodiamond (ND) and nanoplatinum (NP), can modulate the activity of TRPV on human primary keratinocytes, suggesting their potential as a possible pain modulator [1]. CD4+ T lymphocytes also express TRPV channels, and we sought with special interest to examine the effect of DPV576 in modulating the functions of TRPV channel expression and secretion of cytokines on human CD4+ T lymphocytes. Human primary CD4+ T cells were activated with anti CD3/CD28 with and without DPV576 at 1:25 and 1:100 dilutions for 24 hours in vitro. TRPV Receptor expression (TRPV1 and TRPV4) on CD4+ T cells was examined by flow cytometry. The capacity of DPV576 to modulate the activity of TRPV1 agonist capsaicin in CD4+ T cells was also determined. Activation of CD4+ T cells was determined by production of cytokines TNF-α, IFN-γ, and IL-10 using specific ELISA kits. DPV576 treatment of CD4+ T cells that were activated with anti CD3/CD28, resulted in increased expression of TRPV1 channel but had no effect on TRPV4. This was accompanied by increased secretion of IFN-g and reduced expression of TRPV1 in capsaicin activated CD4+ T cells. In addition, DPV576 inhibited the capsaicin, induced the production of both IFN-g and TNF-α, and enhanced the secretion of IL-10. We conclude that short term exposure to DPV576 inhibits the activity of TRPV1 channels in CD4+ T lymphocytes, which may suggest its possible beneficial use for pain management. 

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Efficient HIV-1 Trans Infection of CD4+ T Cells Occurs in the Presence of Antiretroviral Therapy

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz253 ◽

2019 ◽

Vol 6 (7) ◽

Author(s):

Giovanna Rappocciolo ◽

Nicolas Sluis-Cremer ◽

Charles R Rinaldo

Keyword(s):

T Cells ◽

B Cells ◽

Antiretroviral Therapy ◽

T Lymphocytes ◽

Cd4 T Cells ◽

Ex Vivo ◽

Antiretroviral Drugs ◽

Trans Infection ◽

Hiv 1

Abstract Background Antiretroviral therapy (ART) has dramatically improved the quality of life of people with HIV-1 infection (PWH). However, it is not curative, and interruption of ART results in rapid viral rebound. Cell-to-cell transfer of HIV-1, or trans infection, is a highly efficient mechanism of virus infection of CD4+ T cells by professional antigen-presenting cells (APCs), that is, dendritic cells (DCs), macrophages, and B lymphocytes. Methods APC from HIV seronegative donors treated with ART in vitro (CCR5 agonist, NRTI, PI and NNRTI, alone or in combination), were loaded with HIV R5-tropic HIVBal and mixed with autologous or heterologous CD4+ T lymphocytes to assess trans infection. Ex vivo APC from chronic HIV-infected MACS participants before and after initiation of ART, were also loaded with HIV R5-tropic HIVBal and tested for trans infection against autologous or heterologous CD4+ T lymphocytes. Virus replication was measured by p24 ELISA. Results Here we show in vitro that antiretroviral drugs did not block the ability of DCs and B cells to trans-infect CD4+ T cells, although they were effective in blocking direct cis infection of CD4+ T cells. Moreover, ex vivo DCs and B cells from ART-suppressed PWH mediated efficient HIV-1 trans infection of CD4+ T cells, which were resistant to direct cis infection. Conclusions Our study supports a role for HIV-1 trans infection in maintenance of the HIV-1 reservoir during ART.

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Emoxipine Modulates Concentration-Dependent Effects of Cytarabine and Cyclocytidine on Activation of Human T Cells

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2021/v33i59b34376 ◽

2021 ◽

pp. 249-260

Author(s):

Darya B. Nizheharodava ◽

Eugenii I. Kvasyuk ◽

Marina M. Zafranskaya ◽

Aliaksei G. Sysa ◽

Tatyna N. Zhukovets ◽

...

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Cd4 T Cells ◽

Mononuclear Cells ◽

Dependent Manner ◽

Peripheral Blood Mononuclear ◽

Normal Peripheral Blood ◽

Adaptive Immune Cells ◽

Human T Cells

Title: Emoxipine modulates concentration-dependent effects of cytarabine and cyclocytidine on activation of human T cells. Introduction: Both cytarabine and cyclocytidine are used in the treatment of acute myeloid leukemia. Well known that cytarabine and other related cytosine-based nucleoside analogues are being toxic to tumor cells by increasing levels of cellular oxidative stress as it could be abrogated by antioxidants. However, very little is known both about both the effects of combinations of antimetabolites with antioxidants on the cytotoxic innate and adaptive immune cells and whether lymphocytes toxicity affects its anticancer efficiency. Aim: To estimate effects of cytarabine and cyclocytidine with emoxipine on in vitro activated human T cells at concentrations reached during in vivo treatment with high doses, conventional doses and low doses. Materials and Methods: T cells derived from blood donors were activated in vitro in cell culture medium alone or supplemented with cytarabine 0.1-10.0 μM or cyclocytidine 0.1-10.0 μM. Cell characteristics were assessed by flow cytometry. Results: Only cytarabine 1.0-10.0 μM had both antiproliferative and proapoptotic effects. Additionally, these cytarabine concentrations increased the γIFN-producing by CD3+CD4+ T cells and did not affect the release of this cytokine by CD3+CD8+ T cells. In contrast, the lowest concentration (0.1 μM) did not have or showed minor antiproliferative or cytotoxic effects, did not alter the release of γIFN. Cyclocytidine did not affect viability of normal peripheral blood mononuclear cells but decreased the proliferative capacity of activated normal T cells in dose-dependent manner. Additionally, cyclocytidine altered the percentage of γIFN-producing proliferative CD3+CD8+ cytotoxic T cells for any concentration tested (0.1, 1.0, 1 and 10.0 μM) meanwhile highly suppressed the number of the whole amount of CD3+CD8+ cells and did not affect the release of cytokines by CD3+CD4+ T cells. The study of the expression of the CD107a marker showed a significant stimulating effect of 10 µm of citarabine on the activation of subpopulations of T-lymphocytes (CD3+) and cytotoxic T-lymphocytes (CD3+CD8+).

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